Curcumae Rhizoma is a Chinese medicinal herb that is contraindicated during pregnancy. Cold-congelation and blood-stasis are corresponding syndromes to Curcumae Rhizoma. Whether syndrome-based treatment is associated with developmental neurotoxicity of Curcumae Rhizoma remains to be unclear. To verify the theory of traditional Chinese medicine of "syndrome-based treatment during pregnancy", the present study induced the mice blood stasis model by immersing mice in ice water. Pregnant C57 BL/6 wild type(WT) mice and pregnant Nrf2 knock out(KO) mice were randomly divided into control groups and Rhizoma Curcumae exposure groups. The mice were exposed to Rhizoma Curcumae during day 5 to day 18 after pregnancy. The neurodevelopment was examined to evaluate the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice exposed to Rhizoma Curcumae. caspase-3 and caspase-9 activity in brain of the offspring were measured by colorimetric assays. Bcl-2 mRNA and protein expression in brain of the offspring were examined by Real-time RT-PCR and Western blot, respectively. According to the findings, C57 BL/6 mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) had a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring, compared with the normal control group, but with no significant change in those of blood-stasis pregnant mice offspring. However, mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) showed no change in Bcl-2 gene expression and p38 MAPK phosphorylation in brain of the offspring. Nrf2 gene knockout using CRISPR/Cas9 resulted in a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring. In conclusion, developmental neurotoxicity of the blood-stasis pregnant mice exposed to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Nrf2 activation involved in the phenomenon of Rhizoma Curcumae of "syndrome-based treatment during pregnancy", but the upstream signal pathway mechanism value shall be further investigated.
Animals ; Apoptosis ; Brain ; drug effects ; Caspases ; genetics ; Curcuma ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Maternal Exposure ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-E2-Related Factor 2 ; genetics ; Pregnancy ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Random Allocation ; Rhizome ; chemistry ; Signal Transduction

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.
Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; toxicity ; Apoptosis ; drug effects ; Caspases ; genetics ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dioscoreaceae ; chemistry ; Heterografts ; drug effects ; growth & development ; Humans ; Inhibitory Concentration 50 ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Nude ; Phosphorylation ; drug effects ; Plant Tubers ; chemistry ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Saponins ; isolation & purification ; pharmacology ; toxicity

OBJECTIVE: To determine whether chloroquine (CQ), an often used inhibitor of late autophagy and autophagosome/lyosome fusion, can inhibit proliferation of renal carcinoma cells and investigate its effect on sunitinib (ST)-induced apoptosis. METHODS: Renal carcinoma cell line 786 O and ACHN had been used as cellular model and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was carried out to detect the cell viability in response to CQ or ST treatment. Both transmission electron microscope and immunoblotting had been employed to observe apoptotic and autophagic process. To examine the involvement of autophagy in ST-dependent apoptosis, autophagy had been inhibited either chemically or genetically via utilizing autophagy inhibitor or specific small interference RNA (siRNA) targeted to either Ulk1 (unc-51-like kinase 1) or LC3 (microtubule associated protein 1 light chain 3 fusion protein), two essential autophagic proteins. RESULTS: Both ST and CQ induced cell viability loss, indicating that either of them could inhibit renal cancer cell proliferation. Clone formation experiments confirmed the aforementioned results. Furthermore, the combined ST with CQ synergistically promoted the loss of cell viability. By transmission electron microscopy and immunoblotting, we found that the ST induced both autophagy and caspase-dependent apoptosis. While 3-MA, an early autophagy inhibitor, reduced the ST-induced cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), a substrate of caspase 3/7 and often used marker of caspase-dependent apoptosis, CQ promoted the ST-dependent PARP-1 cleavage, indicating that the early and late autophagy functioned differentially on the ST-activated apoptotic process. Moreover, the knock down of either Ulk1 or LC3 decreased the ST-caused apoptosis.Interestingly, we observed that rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin) and an inducer of autophagy, also showed to inhibit cell viability and increased the cleavage of PARP-1 in the ST-treated cells, suggesting that autophagy was likely to play a dual role in the regulation of the ST-induced apoptosis. CONCLUSION ST activates both apoptotic and autophagic process in renal carcinoma cells. Although autophagy precedes the ST-induced apoptosis, however, early and late autophagy functions differentially on the apoptotic process induced by this compound. Additionally, ST can coordinate with the inducer of autophagy to inhibit the cell proliferation. Further research in this direction will let us illuminate to utilize CQ as a potential drug in the treatment of renal carcinoma.
Animals ; Antineoplastic Agents/pharmacology* ; Antirheumatic Agents/pharmacology* ; Apoptosis/drug effects* ; Autophagy/drug effects* ; Caspases ; Cell Line, Tumor ; Chloroquine/pharmacology* ; Kidney Neoplasms/drug therapy* ; Sunitinib/pharmacology*

OBJECTIVE: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells. METHODS: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair. RESULTS: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. CONCLUSIONS The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
Apoptosis ; Caspases/metabolism* ; Cell Proliferation ; Cell Survival/drug effects* ; Doxorubicin/pharmacology* ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Membrane Potential, Mitochondrial ; Oligonucleotide Array Sequence Analysis ; Oxidative Stress/drug effects* ; Phosphorylation ; Propidium/chemistry* ; RNA, Small Interfering/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Rhodamines/chemistry* ; Signal Transduction/drug effects* ; Transcriptome ; Tumor Suppressor Protein p53/metabolism* ; Vitreoretinopathy, Proliferative/drug therapy*

OBJECTIVETo investigate the effect of magnolol on proliferation and apoptosis of HL-60 cells and its mechanism.

METHODSMTT assay was used to measure the proliferation of HL-60 cells after treatment with different concentration of magnolol (5, 10, 20, 40, 80 and 160 µg/ml). The morphological changes of HL-60 cells were examined by light microscopy, and DAPI staining was performed to observe the nuclear morphology of HL-60 cells. The early cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI double-staining. RT-PCR was carried out to examine the mRNA expression of BAX and BCL-2. Western blot was performed to detect the protein expression of caspase family.

RESULTSThe magnolol inhibited HL-60 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time- dependent manner (P < 0.05). HL-60 cells became small, even apoptotic bodies appeared after treatment with magnolol. In addition, nuclear condensation or fragmentation could be observed, which is the typical morphological features of apoptosis. When HL-60 cells were treated with 40 µg/ml of magnolol for 24 h, the ratio of early apoptotic cells reached to (11.7 ± 2.4) %, which was significant different from control (1.4 ± 1.1) % (P < 0.05). RT-PCR results showed that treatment of HL-60 cells with magnolol up-regulated the expression of BAX, whereas down-regulated the expression of BCL-2. Western blot results showed that the cleavages of caspase-3, -8 and -9 were significantly enhanced by magnolol.

CONCLUSIONThe magnolol can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through up-regulation of BAX, down-regulation of BCL-2 and the activation of caspases.

Apoptosis ; drug effects ; Biphenyl Compounds ; pharmacology ; Caspases ; metabolism ; Cell Proliferation ; drug effects ; Down-Regulation ; Flow Cytometry ; HL-60 Cells ; drug effects ; Humans ; Lignans ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.

METHODSNeuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.

RESULTSCd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).

CONCLUSIONCd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

Animals ; Apoptosis ; drug effects ; Cadmium ; toxicity ; Caspases ; metabolism ; Mitochondria ; drug effects ; Neurons ; drug effects ; PC12 Cells ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; metabolism

Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.
Animals ; Apoptosis/*drug effects ; Cadmium/*toxicity ; Caspases/metabolism ; Environmental Pollutants/*toxicity ; Osteoblasts/*drug effects/metabolism ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo confirm the anticancer effect of total annonaceous acetogenins (TAAs) abstracted from Annona squamosa Linn. on human hepatocarcinoma.

METHODSThe inhibitory effect of TAAs was demonstrated in H22-bearing mice. The potency of TAAs was confirmed as its 50% inhibiting concentration (IC50) on Bel-7402 cell under Sulfur Rhodamine B staining. Both underlying mechanisms were explored as cellular apoptosis and cell cycle under flow cytometry. Mitochondrial and recipient apoptotic pathways were differentiated as mitochondrial membrane potential under flow cytometry and caspases activities under fluorescence analysis.

RESULTSThe inhibitory rate of TAAs in mice was 50.98% at 4 mg/kg dose. The IC50 of TAAs on Bel-7402 was 20.06 µg/mL (15.13-26.61µg/mL). Effective mechanisms of TAAs were confirmed as both of arresting cell cycle at G1 phase and inducing apoptosis dose- and time-dependently. Mitochondrial and recipient pathways involved in apoptotic actions of TAAs.

CONCLUSIONTAAs is effective for hepatocarcinoma, via inhibiting proliferation and inducing apoptosis.

Acetogenins ; chemistry ; pharmacology ; therapeutic use ; Animals ; Annona ; chemistry ; Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Organ Specificity ; drug effects ; Spleen ; drug effects ; Thymus Gland ; drug effects ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effects of different concentrations of paraquat (PQ) poisoning on the expression of voltage-dependent anion channel (VDAC) and caspase family in the mitochondria of rat lung tissue, and to explore possible mechanisms of acute lung injury induced by acute PQ poisoning.

METHODSTwo hundred healthy adult Wister rats with equal numbers of male and female ones were randomly and equally divided into control group and poisoned group. The control group received one-time gastric lavage with 1 ml of normal saline, and the poisoned group with PQ (50 mg/kg) diluted in 1 ml of normal saline. Twenty rats were collected at 1, 24, 72, 120, and 168 h after lavage with normal saline or PQ and dissected after anesthesia. Mitochondria were separated from rat lung tissue, and the content of VDAC and caspase-3, -8, and -9 were determined.

RESULTSThe expression of VDAC and caspase-3, -8, and -9 in the poisoned rats were significantly higher than that in the control group (P < 0.001). At 1, 24, 72, 120, and 168 h after exposure, acute diffuse damages were found in alveolar capillary endothelial cells, alveolar epithelial cells, and pulmonary interstitial cells. Inflammatory cell infiltration in the pulmonary interstitium, alveolar structural disorder, and substantially increased fibroblasts were also found in rat lung tissue.

CONCLUSIONPQ poisoning can up-regulate the expression of VDAC and caspase-3, -8, and -9 in mitochondria of rat lung tissue to induce acute lung injury.

Acute Lung Injury ; chemically induced ; pathology ; Animals ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Caspases ; metabolism ; Female ; Lung ; drug effects ; pathology ; Male ; Mitochondria ; drug effects ; metabolism ; Paraquat ; poisoning ; Rats ; Rats, Sprague-Dawley ; Voltage-Dependent Anion Channels ; metabolism

OBJECTIVETo investigate the effects and underlying mechanism of notoginsenoside R1 on amyloid-β (1-42) (Aβ(1-42)) induced mitochondrial apoptotic death in SH-SY5Y cells.

METHODCell viability was assayed by MTT, apoptotic rates were analyzed with PI/Annexin V flow cytometry, Bax and Bcl-2 expression were detected with Western blotting, enzymatic activity of caspase-3, caspase-8 and caspase-9 were measured by ELISA assay.

RESULTThe 6.25-100 nmol x L(-1) of notoginsenoside R1 attenuate Aβ(1-42) induced apoptotic death of SH-SY5Y in dose dependent manner. The ratio of Bcl-2/Bax was elevated in SH-SY5Y with notoginsenoside R1 treatment. Caspase-3 and caspase-9 were activated with notoginsenoside R1 treatment while caspase-8 was not affected.

CONCLUSIONNotoginsenoside R1 could protect SH-SY5Y cells from Aβ(1-42) induced apoptosis via mitochondria related apoptotic pathway.

Amyloid beta-Peptides ; antagonists & inhibitors ; Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cytoprotection ; Ginsenosides ; pharmacology ; Humans ; Mitochondria ; drug effects ; Peptide Fragments ; antagonists & inhibitors